Translational potential of ginsenoside Rb1 in managing progression of osteoarthritis

92 and mineralized nodules, and the highest levels of mRNA expression of osteogenesisrelated genes collagen-1, ALP and BMP-2 in the osteoblasts. The in vivo studies found that the rats in the 1 h group, 1.5 h group and 2 h group had significantly higher bone mineral densities (BMD) than those of the control group, and the highest BMD was obtained in the 1.5h group. The rats in the 0.5 h group and 3h group were not statistically different in the BMD values with the control. The mechanical tests and bone morphometrical analysis results revealed a similar tendency. The serum osteocalcin level was the highest while the TRAP5b level was the lowest in the 1.5 h group. Discussion and Conclusion: Our data has demonstrated that SEMF treatment at 1.5 h per day has the strongest osteogenic activity than other duration times in the rat primary osteoblasts. The in vivo studies provided further supports that SEMF treatment at 1.5 h per day increased the BMD of growing rats to the greatest extent compared to other durations within 3 h. Our study suggests that the osteogenic effects of SEMFs are duration-dependent and 1.5h per day is the optimal duration for improving peak bone mass and may be used for the prevention and treatment of osteoporosis. http://dx.doi.org/10.1016/j.jot.2016.06.073

147 H19 PROMOTES OSTEOGENIC DIFFERENTIATION BY FUNCTIONING AS A COMPETING ENDOGENOUS RNA Jin-fang Zhang a,b, Yuxin Sun a,b, Liangliang Xu a,b, Gang Li a,b a Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, PR China b Stem Cells and Regenerative Medicine Laboratory, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, PR China Objective: Long non-coding RNAs (lncRNAs), extensively transcribed from the mammalian genome, have gained widespread attention in recent years. They serve as important and powerful regulators of various biological activities and play critical roles in a variety of disease progression including differentiation. More and more emerging evidence has demonstrated that some lncRNAs play important regulatory roles in osteoblast differentiation of MSCs, suggesting a potential therapeutic strategy for bone formation. The lncRNA H19, one of the most well-known imprinted genes, is located on human chromosome 11 and it is transcribed only from the maternally inherited allele. Recent researches have highlighted H19 as an active modulator in embryonic placental growth and skeletal muscle differentiation. However, unfortunately, the role of H19 in osteoblast differentiation is largely unknown and its function remains to be characterised. Methods: Cultures of bone marrow-derived MSCs were established from a healthy donor. The gene encoding human H19 was amplified and cloned into a pBABE retrovirus vector. The H19 overexpression stable hMSCs were generated using retrovirus-mediated gene delivery method as previously described. Osteogenic differentiation was induced according to the published protocols and examined by using ALP activity assay, Alizarin Red Staining, and marker genes expression. Furthermore, the in vivo effect of H19 on osteogenesis was evaluated by ectopic bone formation carried out in nude mice. Using bioinformatics tool, the candidate miRNAs targeting were screened out and the direct interaction between H19 and miRNA was identified by using a luciferase activity assay. Results: In the present study, lncRNA H19 was found to be upregulated during osteogenesis in human mesenchymal stem cells. Stable expression of H19 significantly accelerated in vivo and in vitro osteoblast differentiation. Meanwhile, by using bioinformatic investigations and RNA immunoprecipitation assays combined with luciferase reporter assays, we successfully demonstrated that H19 functioned as a miRNA sponge for miR-141 and miR-22, both of which were negative regulators of osteogenesis. Further investigations revealed that H19 antagonized the endogenous functions of these two miRNAs and led to the de-repression of their shared target gene b-catenin, which eventually activated the Wnt/b-catenin pathway and hence potentiated osteogenesis. In addition, we also identified a novel regulatory feedback loop between H19 and its encoded miR-675-5p. miR-675-5p was found to directly target H19 and counteracted osteoblast differentiation. Conclusion: Taken together, these findings indicate that the lncRNA H19 modulates the Wnt/b-catenin pathway by acting as a competing endogenous RNA, which may help to develop a novel therapeutic strategy for promoting fracture healing and bone regeneration. http://dx.doi.org/10.1016/j.jot.2016.06.074

148 Smad7 PARTIALLY KNOCKOUT MOUSE: A NEW ANIMAL MODEL OF OSTEOARTHRITIS Sien Lin a,b,c, Wayne Yuk Wai Lee a,b, Jinfang Zhang a,b, Liao Cui c, Gang Li a,b a The Chinese University of Hong Kong-Astronaut Centre of China (CUHK-ACC) Space Medicine Centre on Health Maintenance of Musculoskeletal System, Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong 518063, China

Session: Disease & Treatment b Department of Orthopaedics and Traumatology, The Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China c Department of Pharmacology, Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, Guangdong, China

Objective: Animal models of osteoarthritis (OA) are of considerable importance in elucidating the underlying mechanisms of joint damage and providing proof of concept in the development of pharmacological and biological agents that may modify structural damage in the OA joint. Currently, there is still a lack of an appropriate small animal model of OA which represents the underlying mechanisms. Since transforming growth factor-beta/Smad (TGF-b/Smad) signalling pathway has been identified as a key pathway in osteoarthritis (OA) initiation and progression, we then hypothesise that animal models of OA could be established by the interruption of TGF-b/Smad signalling. This study aims to investigate the role of Smad7, one of the TGF-b/Smad signalling pathway inhibitors, in the initiation and progression of OA, then further to evaluate whether Smad7 partial knockout mouse is a good animal model of OA. Methods: The genetically engineered Smad7DE1 (KO) and wild type (WT) mice (n Z 15) at the age of 6, 12, or 24 months were terminated for histological analysis. The anterior cruciate ligament transection (ACLT) or sham operation were performed in both the 6-month old Smad7 KO (nZ16) and WT (nZ16) mice. Histology, immunohistochemistry (IHC), and micro-computed tomography (CT) analysis were performed to determine the pathological changes in the articular cartilage and subchondral bone after 6 weeks. The knee joints were harvested and subject to histology and IHC examinations. Results: Histological staining showed that there was no significant difference in the articular cartilage between Smad7 KO and WT mice at 6, 12 or 24 months old. However, cartilage hypertrophic markers (MMP13 and Col X) were significantly upregulated in the intact Smad7 KO mice at 6-months, indicating Smad7 is essential for cartilage homeostasis. In the ACLT surgery model, six weeks after surgery, typical OA phenotype characterised by cartilage destruction, osteophyte formation, and synovium inflammation were all found in the Smad7 KO mice, where only mild degenerative changes were seen in the wild type control mice. Results of Micro-CT showed total bone volume and bone mineral density of subchondral bone were significantly increased in the Smad7 KO mice comparing to the wild type mice after ACLT, indicating a bone hardening in the subchondral bone area. Results of IHC also showed osteogenic marker Osterix was significantly upregulated in the Smad7 KO mice after ACLT, suggesting enhanced bone formation. Conclusion: Smad7 plays an important role in cartilage homeostasis. Lack of Smad7 may contribute to OA intiation. Smad7 KO mice are susceptible to OA progression under mechanical instability conditions. Smad7 KO mice may be used as an animal model of osteoarthritis to further study the underlying mechanisms. http://dx.doi.org/10.1016/j.jot.2016.06.075

152 TRANSLATIONAL POTENTIAL OF GINSENOSIDE Rb1 IN MANAGING PROGRESSION OF OSTEOARTHRITIS Chen Yuanfeng a,b, LinSien a,b, Sun Yuxin a,b, Pan Xiaohua c, Xiao Liubin d, Zou Liyi e, Ho Ki Wai a, Li Gang a,b,f a Department of Orthopaedics & Traumatology, Li Ka Shing Institute of Health Sciences and Lui Che Woo Institute of Innovative Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, PR China b The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, PR China c Department of Orthopaedics and Traumatology, Bao-An District People’s Hospital, Shenzhen, PR China d People’s Hospital of New District Longhua, Shenzhen, PR China e Department of Pharmacology, Guangdong Medical University, Dongguan, PR China f Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China Background: Osteoarthritis (OA) is the most common degenerative joint disorder. Inflammatory cytokines play an important role in OA progression. Previous studies have demonstrated that ginsenoside Rb1 would prevent inflammation and apoptosis in chondrocytes. However, we did not find any animal study that reported the effect of Rb1 on attenuation of the severity of osteoarthritis. Objective: In this study, we used a rat anterior cruciate ligament transection plus medial meniscus resection (ACLT+MMx) model of OA and cell model to investigate whether administration of ginsenoside Rb1 may attenuate the progression of arthritis. Methods: In the in vivo study, the 16-week-old male Sprague-Dawley rats were divided into three groups: Group 1: sham control group; Group 2: Rb1-treated

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group; Group 3: OA Group. In group 2e3, osteoarthritis was induced in the right knee joint with ACLT+MMx in rats. Group 2 received continuous infusion of the ginsenoside Rb1 via an osmotic mini-pump, implanted subcutaneously. At four weeks after treatment, the rat was sacrificed. Interleukin-1 beta (IL-1b) level was evaluated by ELISA; cartilage damage was assessed via histology (Safranin-O/fast green stain) and immunohistochemistry (MMP13, Col X). In the cell study, C5.18 (Rat chondrocyte cell line) was used. The effect on Rb1 of IL-1b- induced MMP13 or Col X expression level in C5.18 cells was investigated. Results: In the in vivo study, characteristics of OA were present in the OA group, contrary to the Rb1 treatment group where, in general, less severe damage occurred: initially, IL-1b level was significantly decreased; then, cartilage degeneration was attenuated, by lower histologic damage scores and the percentage of MMP13 or Col X positive chondrocytes. In the cell study, the results showed that Rb1treatment would relieve the MMP13 or Col X expression in C5.18 cells in IL-1b induction. Conclusion: In the present study, we demonstrated that Rb1 can attenuate the progression or severity of arthritis via reducing the inflammation level. http://dx.doi.org/10.1016/j.jot.2016.06.076

Discussion and Conclusions: mTORC1 signalling has emerged as a critical regulator of bone formation; however, results from in vitro and in vivo studies on the function of mTORC1 in osteoblast lineage are inconsistent. Using mTORC1 specific inhibitor and conditional knockout cell and mouse models, we elucidated the role of mTORC1 in osteoblast formation. Here, we report that activation of mTORC1 is required for preosteoblast proliferation; however, inactivation of mTORC1 is essential for their differentiation and maturation. Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and down-regulation of Runx2. In conclusion, this study clarified the potential role of mTORC1 signalling in the regulation of preosteoblast proliferation and differentiation and identified Notch signalling and Runx2 as critical downstream mediators. Pharmaceutical coordination of the pathways and agents in preosteoblasts may be beneficial in bone formation. http://dx.doi.org/10.1016/j.jot.2016.06.078

163 ASSOCIATION BETWEEN ABO BLOOD GROUP AND PRIMARY KNEE OSTEOARTHRITIS: A CASE-CONTROL STUDY

Tengteng Wang a, Yan Chen a, Jinlong Li a, Qiang Li a, Lianping Xing a, Yongjun Wang b,c, Qi Shi a, Hao Xu a, Qianqian Liang a a Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China b Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA c Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY 14642, USA

Yue Ding, Changchuan Li, Chi Zhang, Guangtao Fu, Shixun Li, Junxiong Qiu, Wei Liu Department of Orthopaedic Surgery, Sun Yat-sen Memorial Hospital, Guangzhou, China Background: Recent studies suggest association between ABO blood group and inflammation, which was crucial in the pathological process of primary knee osteoarthritis. This study was aimed to investigate whether ABO blood group was associated with primary knee osteoarthritis. Methods: We performed a retrospective review of patients of primary knee osteoarthritis as the case group, and a random sampling of healthy blood donors as the control group. The severity of knee osteoarthritis at the first outpatient visit was evaluated by the Kellgren/Lawrence scoring system. Further study was performed to investigate the expression of blood group antigens in synovial tissue of the knee in both cases and controls. Results: 1126 cases and 30299 controls were involved. Logistic regression revealed that AB blood group was a risk factor of primary knee osteoarthritis (PZ0.025 and 0.048 for univariate and multivariate analysis, respectively), independent of age (PZ0.973) and sex (PZ0.520). Patients of blood group AB had a higher K/L score (PZ0.017). Immunohistochemical study indicated association between LeY antigen and primary knee osteoarthritis (PZ0.029). Discussions and Conclusions: This study suggested that blood group AB was associated with primary knee osteoarthritis, as well as its radiological severity. Further study indicated that LeY antigen, which was related to blood group, was associated with primary knee osteoarthritis. http://dx.doi.org/10.1016/j.jot.2016.06.077

169 mTORC1 PREVENTS PREOSTEOBLAST DIFFERENTIATION THROUGH THE Notch SIGNALLING PATHWAY Bin Huang, Dadi Jin, Xiaochun Bai Academy of Orthopaedics, Guangdong Province, The Third Affiliated Hospital of Southern Medical University, China Introduction: Disruption of the balance between bone formation and resorption results in loss of bone mass and causes bone diseases such as osteoporosis. Current therapies for osteoporosis are limited to antiresorptive agents, while bone diseases due to reduced osteoblast activity, such as senile osteoporosis, urgently require targeted treatment and novel strategies to promote bone formation. mTORC1 has emerged as a critical regulator of bone formation and is therefore a potential target in the development of novel bone-promoting therapeutics. Identifying the detailed function of mTORC1 in bone formation and clarifying the underlying mechanisms may uncover useful therapeutic targets. Subjects and Methods: We treated osteoblasts and mice with rapamycin (the mTORC1-specific inhibitor) and established conditional Tsc1 knockout cell and mouse models (with activated mTORC1). We detected the proliferation rate and differentiation potential of osteoblasts with impaired or activated mTORC1 and explored the regulatory mechanism responsible. Results: mTORC1 is activated during preosteoblast proliferation but is suppressed during their differentiation. Inactivation of mTORC1 prevents preosteoblast proliferation but enhances their differentiation in vitro and in vivo. mTORC1 activation in preosteoblasts produces immature woven bone in mice due to excess proliferation and impaired differentiation. mTORC1 inhibits Runx2 expression by activating Notch signalling in preosteoblasts. Preosteoblasts with hyperactive mTORC1 re-acquired the capacity to fully differentiate and mature when subjected to inhibition of the Notch pathway.

174 ANTI-INFLAMMATORY EFFECT OF JUANBI TANG ON TNF-Tg MICE THROUGH PROMOTING LYMPHATIC DRAINAGE FUNCTION

Introduction: Previously, we reported that sufficient lymphatic drainage was favourable for the treatment of Rheumatoid arthritis (RA) and treatment targeting joint lymphatic drainage function contributed to the improvement of chronic inflammation of joints. JuanBi Tang (JBT), a Chinese patent medicine, has been widely used for the treatment of RA in China, which can relieve symptoms of arthritis and activate joint function of patients. The aim of this study is to investigate whether JBT could attenuate inflammation and promote lymphatic drainage function and lymphangiogenesis of TNF-Tg mice. Subjects and Methods: Three-month old TNF-Tg mice and WT littermates were used. (1) The effect of JBT on lymphatic drainage function was detected with Indocyanine green near-infrared (ICG-NIR) lymphatic imaging system. (2) The effect of JBT on ankle joints inflammation were assessed by haematoxylin and eosin (H&E) staining, Alcian blue/orange G (ABHO) staining, and tartrate resistant acid phosphatase (TRAP) staining. (3) The effect of JBT on lymphangiogenesis at inflammatory ankle joints was detected by using anti-LYVE-1 and anti-podoplanin antibodies for double immunofluorescence staining. (4) The effect of JBT on lymphangiogenesis was determined by a zebrafish screening system. (5) The effect of JBT on iNOS expression and NO production of lymphatic endothelial cells (LECs) were also tested. Results: Decreased clearance and pulse from ICG-NIR detection indicated TNF-tg mice lymph function is impaired, but it was rescued by JBT (p<0.05). According to HE staining of ankle sections, JBT (1.2kg/L) could increase TNF-tg mice astragalus bone area (1.540.78mm2) in contrast to saline mice (0.750.24 mm2, p<0.05), almost equal to WT mice (1.580.75 mm2, p>0.05). The inflammation area around the astragalus bone of TNF-tg mice (1.070.44 mm2) increased compared with the WT littermates (0, p < 0.05). JBT could reduce the inflammation area (1.770.44mm2, p<0.05). As to Alcian blue/orange G (ABHO) stain, cartilage area of the astragalus bone (1.770.44mm2, p<0.05) increased more than 20fold in the JBT group compared with the saline group. JBT promotes lymphangiogenesis in the ankle joint of TNF-Tg mice. JBT could also promote the formation of the lymphatic thoracic duct of zebrafish (48h and 72h, p<0.05). In addition, JBT blocked iNOS expression and NO production of LECs. Conclusion: JBT decreases synovial inflammation, bone erosion and cartilage erosion, and promotes lymphatic drainage function and lymphangiogenesis in TNF-Tg mice. JBT is a promising agent for treating Rheumatoid arthritis and promoting the lymphatic drainage function might be one of its mechanisms. http://dx.doi.org/10.1016/j.jot.2016.06.079

228 TANSHINOL-LOADED BONE-TARGETING LIPOSOME ACCELERATES DELAYED FRACTURE HEALING IN MICE Yanzhi Liu a,b, Zhenshan Jia b, Xiang Gao c, Xiaoyan Wang b, Xiaobei Wang b, Liao Cui a, Dong Wang b a Department of Pharmacology, Guangdong Key Laboratory for R & D of Natural Drugs, Guangdong Medical University, China b University of Nebraska Medical Centre, USA c Stem Cell Research and Cellular Therapy Centre, Affiliated Hospital of Guangdong Medical University, China Objective: Bone fracture non-union is a major clinical challenge in orthopaedic practice. In addition to surgical intervention and autologous bone grafts, bone